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Although medical ultrasound offers compelling opportunities to improve therapy in principle, progress in the field has been limited because of an insufficient understanding of the potential genotoxic and cytotoxic effects of ultrasound on biological systems. This paper is mainly focused on an in vitro study of effects with respect to genotoxicity and viability induced by 1- and 3-MHz medical ultrasound in murine fibroblasts (NIH-3T3) at low-intensity exposure (spatial peak temporal average intensity Ita

This work is focused on the in vitro study of the effects induced by medical ultrasound (US) in murine fibroblast cells (NIH-3T3) at a low-intensity of exposure (spatial peak temporal average intensity Ita

Scanning near-field optical microscopy (SNOM) makes it routinely possible to overcome the fundamental diffraction limit of standard (far-field) microscopy. Recently, aperture-based infrared SNOM performed in the spectroscopic mode, using the Vanderbilt University free electron laser, started delivering spatially-resolved information on the distribution of chemical species and on other laterally-fluctuating properties. The practical examples presented here show the great potential of this new technique both in materials science and in life sciences. (C) 2004 Elsevier B.V. All rights reserved.

Vibrational spectroscopy provides a powerful tool to understand the molecular structures. When applied to the liquid water, this technique reveals so many details which can also shed a light on the supramolecular arrangement of the most ubiquitous of the substances. In particular, the two fluid model of water, proposed several decades ago, founds experimental evidence. Moreover, some fundamental parameters calculated in the realm of the theory of Quantum ElectroDynamics applied to liquid water can be actually measured showing an excellent agreement with the theory. This allows to add a dynamical origin to the mixed cluster model of water well known by the biologists for fifty years and opens the way to the dawn of a real quantum biology. © Published under licence by IOP Publishing Ltd.

Structural and functional effects of exposing murine fibroblasts (NIH 3T3) to therapeutic ultrasound at 1 MHz frequency are described. These bioeffects can be attributed to the formation of free radical species by sonolysis of water. When cavitation occurs, dissociation of water vapor into H atoms and OH radicals is observed; these H atoms and OH radicals combine to form H(2), H(2)O(2), and HO(2). The radicals can chemically modify biomolecules, for example enzymes, DNA, and lipids. Generation of free radicals during exposure to ultrasound with or without encapsulated microbubbles (contrast agents) was studied by use of electron paramagnetic resonance with DMPO spin trapping. Recently the potential for possible use of these microbubbles in gene therapy has been investigated, because of the ability of the stabilized microbubbles to release their content when exposed to ultrasound. Structural changes were studied by Fourier-transform infrared spectroscopy, and induction of possible genotoxic damage by exposure of the cells to therapeutic ultrasound at 1 MHz frequency with our experimental device was verified by use of the cytokinesis-block micronucleus assay.

Fourier transform infrared spectroscopy in attenuated total reflection can be used to discriminate the necrotic from the apoptotic cell death in a tumoral T cell line irradiated by a UV source able to induce both apoptosis and necrosis. Using Jurkat cells as the model system, significant spectral differences in the irradiated cells vs. time were observed in the lipid-proteins ratio absorbance band at 1,397 cm(-1) and in lactic acid IR band at 1,122 cm(-1); these spectral features are inversely correlated with the percentage of apoptotic cells assessed by flow cytometry. From the analysis of second derivatives in the IR spectral region between 1,800 and 900 cm(-1), we have detected two significant spectral changes: the first centered at 1,621 cm(-1) by analyzing the components of the amide I band and the second centered at 1,069 cm(-1) due to C-O stretching vibration of the DNA backbone sensitive to the dehydrated state of DNA; these identified differences in the intracellular biomolecules have been allowed to monitor the necrotic process. The variations in the spectral data set have been identified by the Kruskal-Wallis test and confirmed by the hierarchical cluster analysis.

Gold metallodrugs form a class of promising antiproliferative agents showing a high propensity to react with proteins. We exploit here X-ray absorption spectroscopy (XAS) methods [both X-ray absorption near-edge spectroscopy (XANES) and extended X-ray absorption fine structure (EXAFS)] to gain insight into the nature of the adducts formed between three representative gold(I, III) metallodrugs (i.e., auranofin, [Au(2,2'-bipyridine)(OH)(2)](PF(6)), Aubipy, and dinuclear [Au(2)(6,6'-dimethyl-2,2'-bipyridine)(2)(μ-O)(2)](PF(6))(2), Auoxo6) and two major plasma proteins, namely, bovine serum albumin (BSA) and human serum apotransferrin (apoTf). The following metallodrug-protein systems were investigated in depth: auranofin/apoTf, Aubipy/BSA, and Auoxo6/apoTf. XANES spectra revealed that auranofin, upon protein binding, conserves its gold(I) oxidation state. Protein binding most probably takes place through release of the thiosugar ligand and its subsequent replacement by a thiol (or a thioether) from the protein. This hypothesis is independently supported by EXAFS results. In contrast, the reactions of Aubipy with serum albumin and of Auoxo6 with serum apoTf invariantly result in gold(III) to gold(I) reduction. Gold(III) reduction, clearly documented by XANES, is accompanied, in both cases, by release of the bipyridyl ligands; for Auoxo6 cleavage of the gold-gold dioxo bridge is also observed. Gold(III) reduction leads to formation of protein-bound gold(I) species, with deeply modified metal coordination environments, as evidenced by EXAFS. In these adducts, the gold(I) centers are probably anchored to the protein through nitrogen donors. In general, these two XAS methods, i.e., XANES and EXAFS, used here jointly, allowed us to gain independent structural information on metallodrug/protein systems; detailed insight into the gold oxidation state and the local environment of protein-bound metal atoms was achieved in the various cases.

In this study we have used for the first time the infrared spectroscopy as a tool to reveal the effects induced on a cell line, Jurkat T-lymphocytes, by ultrasound (US) used in therapy at 1 MHz frequency. We carried out the experiments using two different setups allowing to change the acoustic field parameters by varying the position of the sample with respect to the ultrasonic transducer and the sonication time with different duty cycles. The parameters of acoustic field measured by a hydrophone were compatible with the cavitation phenomenon in both setups. Genotoxic damage in exposed cells was ascertained by the cytokinesis-block micronucleus (CBMN) assay whilst cell structural changes due to sonication, were revealed by attenuated total reflection-Fourier transform infrared (ATR-FTIR) spectroscopy, a non-destructive technique that allows, by the acquisition of IR spectra within a few minutes, one to obtain simultaneous information on all cellular macromolecules in a cell population. Infrared spectroscopy results highlight changes of functional groups typical of proteins, nucleic acids and lipids at 118 J/cm 2 energy dose, while the micronuclei test shows no toxic damage to the cellular samples; higher energy doses are necessary to reveal significant toxic effects together with structural changes.

The experiment was performed to prove that exposure to a relatively weak extremely low frequency (ELF) magnetic field retards tadpoles' development.Two cohorts of Xenopus laevis laevis (Daudin) tadpoles were exposed during their immature period ( approximately 60 days) to a 50 Hz magnetic field of 63.9or = Bor = 76.4 microT rms (root mean square, average values) magnetic flux density in a solenoid. At the same time, as controls, two comparable cohorts were reared in two aquariums remote from the solenoid. Cohorts' degree of development was quantified by daily inspections of animal limbs and attributing them to a stage of the Nieuwkoop and Faber ( 1956 ) classification. The experiment was replicated three times.(a) Mean developmental rate of exposed cohorts was reduced with respect to controls (0.43 vs. 0.48 stages/day, p0.001) starting from early larval stages; (b) Exposure increased the mean metamorphosis period of tadpoles by 2.4 days compared with the controls (p0.001); (c) Maturation rates of exposed and control tadpoles changed during maturation period; and (d) Important mortality, malformations or teratogenic effects were not observed in exposed matured tadpoles.A long-term exposure of X. laevis tadpoles to a relatively weak 50 Hz magnetic field causes a sub-lethal effect that slows down their larval developmental rate and delays their metamorphosis.

We have made a preliminary analysis of the results about the effects on tumoral cell line (lymphoid T cell line Jurkat) induced by UVB radiation (dose of 310 mJ/cm(2)) with and without a vegetable mixture. In the present study, we have used two techniques: Fourier transform infrared spectroscopy (FTIR) and flow cytometry. FTIR spectroscopy has the potential to provide the identification of the vibrational modes of some of the major compounds (lipid, proteins and nucleic acids) without being invasive in the biomaterials. The second technique has allowed us to perform measurements of cytotoxicity and to assess the percentage of apoptosis. We already studied the induction of apoptotic process in the same cell line by UVB radiation; in particular, we looked for correspondences and correlations between FTIR spectroscopy and flow cytometry data finding three highly probable spectroscopic markers of apoptosis (Pozzi et al. in Radiat Res 168:698-705, 2007). In the present work, the results have shown significant changes in the absorbance and spectral pattern in the wavenumber protein and nucleic acids regions after the treatments.

A detailed knowledge of the interaction between bacterial membranes and antibiotics provides important information to prevent high levels of antibiotic resistance exhibited by pathogenic strains. We investigated by energy dispersive X-ray diffraction (EDXD) the structure ordering of dioleoyl-phosphatidylcholine (DOPC) lipid interacting with antimicrobial peptide alamethicin, varying the lipid/peptide (L/P) molar ratio under two different hydration levels. In conditions of full hydration (100%) we found that the bilayer thickness is constant between L/P = 20 and L/P = 80 indicating that in this range, the system has reached the threshold value for the channel formation, while at the relative hydration of 45% a linear decrease of the bilayer thickness as function of L/P was revealed. The kinetic study of the complex alamethicin–DOPC at different L/P values, shows that the Bragg peak energy variation versus the hydration time has a biexponential behavior characterized by two different time constants.

We investigated, by FTIR microspectroscopy, the interaction of some cationic and helper lipids with DNA deposited on solid supports allowing the formation of ordered structures. FTIR experiments were performed using both conventional and synchrotron radiation infrared sources and by depositing the lipids on both silicon and wedged diamond plates. Cationic liposomes composed of DOTAP (1,2-dioleoyl-3-trimethylammonium–propane) or DDAB (dimethyldioctadecylammonium bromide) and one helper neutral lipid DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine) at different cationic lipid/DNA weight ratio, were studied. Spectra of the single components are compared with those obtained for both equimolar cationic–neutral mixture and lipid/DNA complexes.

A novel approach to the study of RBCs based on the collection of three-dimensional high-resolution AFM images and on the measure of the surface roughness of their plasma membrane is presented. The dependence of the roughness from several parameters of the imaging was investigated and a general rule for a trustful analysis and comparison has been suggested. The roughness of RBCs is a morphology-related parameter which has been shown to be characteristic of the single cells composing a sample, but independent of the overall geometric shape (discocyte or spherocyte) of the erythrocytes, thus providing extra-information with respect to a conventional morphology study. The use of the average roughness value as a label of a whole sample was tested on different kinds of samples. Analyzed data revealed that the quantitative roughness value does not change after treatment of RBCs with various commonly used fixation and staining methods while a drastic decrease occurs when studying cells with membrane–skeletal alteration both naturally occurring or artificially induced by chemical treatments. The present method provides a quantitative and powerful tool for a novel approach to the study of erythrocytes structure through an ultrastructural morphological analysis with the potential to give information, in a non-invasive way, on the RBCs function.

XANES spectra of non structural protein 3 (NS3) have been calculated using 4 Zn coordination models from three crystallographic structures in the Protein Data Base (PDB): 1DY9, subunit B, 1CU1 subunit A and B, and 1JXP subunit B. Results indicate that XANES is an appropriate tool to distinguish among them. Experimental XANES spectra have been simulated refining crystallographic data. The model obtained by XAS is compared with the PDB models.

Lipid membranes were studied by infrared scanning near-field optical microscopy at several wavelengths and by x-ray reflectivity. Together with the x-ray data, the optical images indicate the formation of locally ordered multiple bilayers, and the topographical micrographs reveal the presence of islands at the surface, both critically important features for biotechnology and medical applications such as biosensors and gene therapy.